1. Field of the Invention
The present invention relates generally to proteolytic enzyme compositions and procedures for digesting connective tissue. More particularly, the present invention is directed to proteolytic enzyme compositions which reliably and reproducibly digest connective tissues in a variety of procedures including therapeutic applications and related cell dissociation and cell isolation techniques.
2. Description of Relevant Art
Proteolytic enzymes have found wide utility in a variety of laboratory and clinical applications. Typically these applications involve cell dissociation and related therapeutic procedures which are benefitted by the ability of proteolytic enzymes to hydrolytically break-up or loosen connective tissue networks. For example, bacterial collagenase derived from Clostridium histolyticum has been used to disperse cells in laboratory tissue culture applications. Additionally, collagenase has demonstrated utility in cell isolation procedures such as those associated with isolating pancreatic islets and dispersing a variety of tumor cells. Other uses for collagenase involve its topical use in clinical applications in which collagenase compositions are applied in the treatment of burns or ulcers and wound healing. Other uses include the treatment of Peyronie's disease and as an adjunct to cryoprostatectomy for the removal of retained cryoslough, intervertebral discolysis, and in ophthalmic surgery.
Like collagenase, chymopapain, the major proteolytic component of the crude latex of Carica papaya, has been utilized in the treatment of abnormal or herniated discs to selectively dissolve the nucleus pulposus of the disc. Other uses associated with chymopapain include its utility in cryosurgical healing processes.
Combinations of proteolytic enzymes such as compositions of bacterial collagenase and hyaluronidase are reportedly particularly useful for digesting or dissolving prostatic tissue in the treatment of benign prostatic hypertrophy. The combination of these two proteolytic enzymes apparently dissolves prostatic tissue in order to relieve the obstructive symptoms of prostatic hypertrophy.
Recently, bacterial collagenase derived from Clostridium histolyticum has found utility in procedures involving the dissociation and isolation of microvessel cells embedded in fatty tissues. These procedures generally involve combining fatty tissues having embedded microvessels, such as liposuctioned fat, with collagenase under conditions which cause the collagenase to disrupt and digest the connective tissue. By carefully separating the cells from the digested tissue, viable microvessel cells are recovered.
These viable and intact microvessel cells have found particular utility as a coating on the interior of synthetic small diameter vascular grafts implanted in humans and animals to replace blood vessels. Similarly, microvessel cells are useful as deposits on the surface of biomedical implant devices in general where they provide an improved biocompatibility to the implant. Apparently the microvessel cells contribute to the prevention of protein deposits and related cellular deposits on the implants which are known to occur when foreign materials are placed in contact with blood and tissue. In the case of vascular grafts these deposits can quickly cause the vessel to occlude, resulting in the functional failure of the graft.
One problem associated with the use of commercial sources of crude collagenase to digest fatty tissues, as well as connective tissue in general, is that the degree to which tissue digests or hydrolyzes is unpredictable. Moreover, cells which are isolated from tissue digestion procedures utilizing crude collagenase can be inferior in quality and have a low degree of viability and efficacy. Even when viable cells are successfully isolated, the yield and degree of viability is unpredictable.
The unpredictable nature of these procedures may be attributed to the lot variations inherent in commercial sources of crude collagenase. Another factor which may contribute to the lack of reproducibility in these procedures is the nature of the mixture of tissues being digested. While connective tissues are formed largely of collagen, for which collagenase is specific in its hydrolytic activity, significant amounts of other proteins and glycoproteins are additionally found in connective tissue matrices. Thus, collagenase alone may not effectively hydrolyze all of the tissue mixtures.
Further, collagenase derived from native bacteria differs widely in its collagen specific hydrolytic activity and the amount and character of impurities, including other proteases and toxins. The protease impurities in crude collagenase contribute to the hydrolysis of minor proteins in connective tissue and actually aid in the digestive process. However, unfortunately protease impurities are active with proteins generally and will react with collagenase, causing the crude collagenase to be subject to catalytic degradation. The toxin impurities associated with crude collagenase can be a serious problem for procedures involving both in vivo and in vitro applications. Toxins can disrupt cell membranes, destroy cell viability and generally lower cell yield. Additionally, impurities can contain variable amounts of bacterial DNA, which may cause cell damage and possible immunological problems when isolated cells or tissue digestion procedures involve in vivo applications. Finally, the noncollagenase impurities found in crude collagenase may act as sensitizing antigens which can cause anaphylactic shock if administered to patients.
Thus, in view of the varying and unpredictable nature of crude collagenase compositions which contain a host of proteolytically active and unreactive compounds as well as toxins, the use of crude collagenase compositions for therapeutic digestion procedures and cell dissociation techniques can be unreliable. Alternatively, using purified collagenase having essentially only collagen specific hydrolytic reactive components in these tissue digestion procedures has not been successful. The failure of purified collagenase in these procedures is apparently due to the tissue containing noncollagen proteins which are not digested by collagen specific collagenase. Crude collagenase will digest these tissues because it contains other proteolytic enzymes. However, it does so to a varying and unpredictable degree.
It has been suggested that bacteria genetically engineered to produce limited forms of collagenase having known molecular weights and hydrolytic activity may be advantageous when utilized in tissue digestion procedures. However, even when a wide range of isomeric forms of collagenase are utilized in tissue digestion procedures, the narrow specificity of collagenase in general precludes effectively hydrolyzing all of the tissue. This is because the wide spectrum of proteolytic activity and noncollagen specificity supplied by crude collagenase derived from native bacteria is not available in these genetically engineered sources of collagenase. Even when toxins are removed from genetically engineered sources of collagenase, the resulting collagenase compositions do not provide hydrolytic characteristics suitable for efficacious tissue digestion and/or cell dissociation procedures.
Accordingly, it is an object of the present invention to provide proteolytic enzyme compositions capable of digesting connective tissue in a reproducible and predictable manner.
It is another object of the present invention to provide proteolytic enzyme compositions capable of dissociating and isolating viable cells with predictable and reproducible yields and quality.
It is another object of the present invention to provide tissue digestion procedures and associated therapeutic procedures which provide reproducible and predictable results.
It is a further object of the present invention to provide viable and efficacious microvessel cells isolated from fatty tissue mixtures for incorporating on the inner surface of artificial vascular grafts and other medical implants.